Reversible, parallel and multitask cloning method and kit

ABSTRACT

The present invention is related to a reversible, parallel and/or multitask cloning method and kit, which improve the cloning of (preferably multiple) genetic element(s) in a nucleic acid construct such as vector or in chromosome of a cell and the rapid and efficient selection of said construct with a correct integration of said genetic element(s) either in vitro or in vivo.

FIELD OF THE INVENTION

The present invention is related to a reversible, parallel and/ormultitask cloning method and kit, which improves the cloning of(preferably multiple) genetic element(s) in a nucleic acid constructsuch as a vector or the chromosome of a cell and the rapid and efficientselection of constructs with a correct integration of said geneticelement (s), either in vitro or in vivo.

BACKGROUND OF THE INVENTION

To obtain complex molecular constructs comprised of multiple geneticelements, the selection of the genetic events (insertion(s) and/ordeletion(s) and/or inversions(s) of DNA fragments) that will cause theassemblage of the target construct comprised of the said geneticelements at the right position and with the right orientation is usuallya time consuming procedure.

In particular, one is necessary faced with the major problem ofselecting different multiple genetic events (insertion, deletion,inversion of a genetic sequence in a nucleic acid construct), possiblyin the same reaction tube.

Therefore, a molecular biologist should usually obtain a genetic event(insertion, deletion, inversion of a genetic sequence in a nucleic acidconstruct) separately and not simultaneously in the same reaction tubeand should avoid any mistake (incorrect integration of a geneticsequence in the wrong direction, etc,) during said genetic manipulation.

AIMS OF THE INVENTION

A first aim of the invention concerns methods and tools which provide asolution to the above-mentioned problems, in particular methods andtools which allow a molecular biologist to insert and/or remove agenetic element, or to obtain a modification in the lecture orientationof said genetic element (inversion) in a nucleotide sequence, either invitro or in vivo.

Another aim of the present invention is to provide methods and toolswhich allow the creation of a genetic construct (such as a vector or thechromosome of a cell), either in vitro or in vivo, and assembled throughthe insertion(s), deletion(s) and/or inversion(s) of multiple geneticelements and the selection of the said genetic construct havingincorporated (deleted or inverted) correctly these genetic elements.

A further aim of the present invention is to provide tools which allow abiologist to perform the step of the method in parallel and to performat the same time multiple tasks (selection of multiple genetic events)in the same reaction tube or not.

A last aim of the present invention is to provide tools which allow thatgenetic events (insertions and deletions and inversions) are reversible,such that any nucleic acid construct can be viewed as a set of elementsthat can be recycled, i.e., re-used for the assemblage of otherdifferent nucleic acid constructs.

SUMMARY OF THE INVENTION

In the method and kit described hereafter, the person skilled in the artuses specific genetic constructs, which are the tools for performing thecloning and selection method according to the invention. Said tools aregenetic constructs that could be integrated in vector(s) (plasmid(s) orvirus(es), including bacteriophage(s)) or in the chromosomal genome of acell suitable for obtaining the cloning and selection of the correctassemblage of various genetic elements. All these methods and systemsallow the assemblage of one or more foreign genetic element(s) (targetsequences of interest) in said nucleic acid construct vector orchromosome of a cell at specific sites. The integration of a foreign(preferably autologous) genetic element the nucleic acid construct ofthe invention could be done by techniques known to the person skilled inthe art such as, but not limited to classical restriction/ligation, sitespecific recombination, TOPO cloning and homologous recombination. Theassemblage of genetic elements can involve insertion(s), deletion(s)and/or inversion(s) of nucleotide sequences. In the method according tothe invention, the selection of correctly inserted sequences is obtainedby using specific markers, which are nucleotide sequences encodingmolecules that are toxic for a cell or molecules which are inhibitors ofsuch toxic molecules and/or block to toxic activity of such moleculesexpressed in the cell. Preferably, said molecules are either poison(s),and/or inhibitor(s) to poison(s), preferably selected from (but notrestricted to) the group consisting of the following poison/antidotesystems: Ccdb/Ccda, Kid/Kis, Hok/Sok, Doc/Phd, RelE/RelB,PasA/PasB/PasC, MazE/MazF, ParE/ParD.

In the method according to the invention, said foreign nucleotideelements are advantageously linked (at its 3′ or 5′ or both ends) to oneor more promoter/operator nucleotide sequences, such as, but not limitedto, constitutive promoters allowing the expression of a targetnucleotide sequence incorporated in the nucleic acid construct accordingto the invention, when they are disposed according to the suitable andrequested lecture orientation.

In the method according to the invention, the person skilled in the artuses suitable cell strain(s) (prokaryotic and/or eukaryotic) which areeither resistant or sensitive to one or more of said toxic molecules inorder to obtain and select recombinant(s). The properties of cellstrains can for example be due to the existence of gene(s) coding forpoison and/or antidotes and integrated in the chromosome(s) of a cell orpresented in episomal sequences such as plasmids.

Reversible Cloning and Selection Method and Kit

A first aspect of the present invention is related to a reversiblecloning method and kit for which several specific preferred examples aredescribed in details hereafter, in reference to the FIGS. 2 to 5.

The elements used in the method of the invention are specific cells anda genetic preferably integrated in a vector or a chromosome of a cellcomprised of either:

-   -   a promoter/activator sequence 11 disposed upstream of a first        and a second nucleotide sequence (1,2) encoding two different        toxic molecules (such as a poison 1 and a poison 2) (FIG. 2,        left), or    -   a first promoter/activator sequence 11 disposed upstream of a        first nucleotide sequence 1 encoding a toxic molecule (such as a        poison 1) and, disposed in the opposite lecture direction of the        first promoter/activator sequence 11, a second        promoter/activator sequence 12 disposed upstream of a second        nucleotide sequence encoding an antidote 2′ to a second toxic        molecule (such as poison 2) (FIG. 3, left), or    -   a promoter/activator sequence 11 disposed upstream of a first        and a second nucleotide sequence (1,2′) encoding, respectively,        a first toxic molecule (such as poison 1) and an antidote to a        second toxic molecule (such as poison 2) different from said        first toxic molecule (FIG. 4, left).    -   The terms “a nucleotide sequence encoding a toxic molecule or an        antidote to a toxic molecule” also include sequences comprising        multiple coding portions encoding several identical toxic        molecules.

The insertion of a foreign target nucleotide sequence (A) “in” or as “areplacement” of the nucleotide sequence (1) encoding a toxic moleculeelement will allow either:

-   -   the inactivation of the nucleotide sequence 1 encoding the first        toxic molecule, plus the activation or maintenance of the        activation of the sequence 2 encoding the second toxic molecule        (FIG. 2); or:    -   the inactivation of the first nucleotide sequence 1 encoding the        first toxic molecule, plus the inactivation of the nucleotide        sequence 2 encoding the antidote to the second toxic molecule        (FIG. 3); or:    -   the inactivation of the first nucleotide sequence 1 encoding the        first toxic molecule (FIG. 4).

The inserted foreign genetic element(s) (target sequence) may be aregulatory sequence or gene(s) of interest (possibly linked to one ormore promoter/operator sequences).

The selection of the genetic event (insertion) can be obtained in a cellstrain sensitive to the first toxic molecule 1 (FIGS. 2&3&4) andpossibly resistant to the second toxic molecule 2 (FIG. 2).

However, the said genetic event (insertion or replacement) is reversiblethrough the replacement of the inserted element (target sequence) by theelement that had been deleted following the recombination and insertionmade in the first step. This reverse reaction deletion of a targetsequence is selected in a strain both resistant to the toxic molecule 1and sensitive to the toxic molecule 2 (FIGS. 2, 3, 4) plus, possibly,producing the toxic molecule 2 (FIGS. 3&4).

This reversible cloning and selection method is also suitable forobtaining an inversion of an integrated genetic element. A specificexample is described in details hereafter, in reference to the FIG. 5.Indeed, the orientation of a sequence of interest can be reversedthrough the method of the invention (preferably following the insertionstep of FIG. 4) or through a direct insertion of the target sequencebetween two different antidote sequences (1′, 2′). Said genetic element(target sequence) associated to a promoter/operator (either at its 3′ or5′ end), is initially integrated between two nucleotide sequences(1′,2′) encoding respectively two different antidotes to two differenttoxic molecules 1 and 2. Said two nucleotide sequences (1′,2′) encodingthe two different antidotes are disposed in opposite lectureorientations (disposed upstream and downstream the target nucleotidesequence in opposite divergent lecture orientation). This constructallows to select for the recombination event(s) which will cause thetarget nucleotide sequence of interest and its associated promoter tohave either the same orientation as the nucleotide sequence 1′ encodingthe first antidote to the first toxic molecule (selection done in astrain both sensitive to and producing poison 1) or the same orientationas the nucleotide sequence 2′ encoding the second antidote to the secondtoxic molecule (selection done in a strain both sensitive to andproducing poison 2). (see WO 02/066657 incorporated herein by reference)

Parallel and/or Multitask Cloning and Selection

The above-mentioned reversible cloning and selection method and elements(nucleic acid construct or vector and specific cells strains) can alsobe used in a parallel and/or multitask cloning and selection methoddescribed hereafter (in details in the following example in reference tothe FIG. 1).

The assemblage of multiple foreign genetic elements (different targetsequences) in the vector or in the chromosome of a cell (either in vitroor in vivo) and the selection of the correct assemblage is obtained bythe use of multiple nucleic acid construct comprising sequences encodingone or more (different or identical) toxic molecules and/or theirantidotes. According to the type of the nucleic acid construct and thetype of selective markers (encoding toxic molecules(s) and/orantidote(s) to toxic molecule(s)), the person skilled in the art canselect the suitable events of insertion(s), deletion(s) and/orinversion(s) applied with said multiple genetic element(s).

Said cloning and selection method may require multiple steps possiblyperformed (sequentially) in the same reaction tube or inside a singlecell.

Said method can be combined with the steps and means for performing invitro protein synthesis (using in vitro transcription and translationkits).

Another aspect of the present invention is related to the algorithms,computer programs, and data bases (comprised of codes and means possiblystored in a computer readable medium) that can assist performing one ormore step(s) of the method according to the invention. Said algorithms,data bases, and program codes means are used to define the correctcombination of (but not limited to):

-   -   suitable markers (encoding the toxic molecule and/or the        antidotes to said toxic molecule);    -   suitable cell strain(s) for selecting the suitable genetic        events;    -   suitable pre-starting nucleic acid construct(s),    -   suitable genetic element(s) (target nucleotide sequences and/or        their operator/promoter sequences) to be inserted, deleted        and/or reversed;    -   reaction mixture (including but not restricted to recombines        mixtures, buffer, media, enzymes, . . . ) that are necessary for        the assemblage/production of the molecular construct.

The algorithms, computer programs, and data bases are also able tocontrol one or more step(s) of the method according to the invention,possibly performed by automate (s).

Another aspect of the present invention is related to kits of parts(cloning and/or selection kits) comprising the suitable elements forperforming the method according to the invention, in particular computerprograms mentioned above, nucleic acid construct(s), cell strain(s)and/or usual products and media used in the cloning and selectiontechniques.

Another aspect of the present invention is related to automates allowingto perform the method according to the invention and using theabove-mentioned kit(s) of parts. Said kit(s) of parts (cloning andselection kits, combined with adequate media, cells and media present invitro transcription and translation kits) and automates could alsocomprise other elements, such as a buffer solutions, pipetingelement(s), primers for genetic amplification, cell culture media andmeans for recording results and for the storage of data.

The present invention will be described in detail in the followingexamples, in reference to the enclosed figures presented as non-limitingillustration of the various aspects of the present invention.

SHORT DESCRIPTION OF THE DRAWINGS

The FIG. 1 is an example of complex genetic construct obtained byparallel and multiple genetic events performed by the method of thepresent invention.

The FIGS. 2 to 5 are examples of reversible cloning and selecting methodand kit according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention allows the making of complex genetic constructionsthrough the use of (i) simultaneous and (ii) parallel events (thevarious recombinations and selection events present almost the samefrequency). The “multitask” nature of the invention is defined asfollows: for example, the invention allows to perform the insertion ofgenetic elements A and C, the deletion of genetic elements E and F, andthe inversion of genetic elements B and D, some or all events (FIG. 1)being performed simultaneously in vitro (i.e., in the same tube) or invivo (i.e., in the same organism). The final product of theabove-mentioned events is a complex construct comprised of the geneticelements A, B, C, and D, all with the same orientation. The simultaneousselection of several genetic events (e.g., here, insertions, deletions,inversions, recombinations) is achieved through the use of a differentselective marker (here poisons and antidotes genes for example) for eachof the events. Filled black arrows represent promoters.

Plasmid 1 is amplified in a strain resistant to poison 1. Plasmid 2 isamplified in a strain resistant to poisons 6 and 9. Plasmid 3 isselected in a strain:

-   -   sensitive to poisons 1 and 6 (for the selection of the insertion        of genetic elements A and C),    -   sensitive to poisons 3 and 5 (for the selection of the        inversions of genetic elements B and D),    -   sensitive to poisons 7 and 8 (for the selection of the deletions        of genetic elements E and F),    -   sensitive to poison 9 (for the selection of the recombination        event between the construct made from plasmid 1 and the        construct made from plasmid 2),    -   and producing poison 3, poison 5, poison 7 and poison 8.

Realization of each “recombination” event can be done through techniquessuch as, but not limited to, classical restriction/ligation,site-specific recombination, or homologous recombination. Specificity ofeach genetic event (insertion, deletion, inversion, etc.) is insured bythe specificity of the recombination event. For example, specificity ofan insertion (both the location of the insertion and the orientation ofthe insert (target nucleotide sequence)) can be achieved by the use ofdifferent DNA sequences bordering both the insertion site and thefragment to be inserted (these DNA sequences can be selected by the manskilled in the art for performing said recombination event). Theseflanking sequences form either different site-specific recombinationelements (in the case of site-specific recombination) or differentelements of homology (in the case of homologous recombination). Thesimultaneous selection of several genetic events (e.g., an insertion, adeletion, and an inversion) is achieved through the use of a differentselective marker for each of the events. As each of the genetic eventsis rare by nature, the selection for the simultaneous presence of allevents requires the use of very efficient selective markers (e.g., butnot limited to, antidote/poison genes).

The parallel cloning nature of the invention is defined as follows: Ndifferent genetic constructs that are produced in the same reaction mix(i.e., in the same tube) through the multitask process described abovecan be pre-designed such that their assemblage (here, the assemblage ofthe construct made from plasmid 1 with the construct made from plasmid2) can be generated through recombination events as well. In otherwords, N−1 genetic constructs can be viewed as donors and 1 construct asa receptor. For example, N constructs can be combined through the use ofn−1 selective markers for the selection of n−1 recombination events(FIG. 1).

Furthermore, the invention allows to use the products of themultitask/parallel cloning process as building blocks of new reactions.Indeed, a construct produced through the invention is a uniquecombination of building blocks that can be re-used for new (anddifferent) constructs; i.e., the process is reversible and extendable,as shown in the FIGS. 2 to 4.

In FIG. 2, the DNA fragment to be inserted encodes for a target sequenceof interest plus a promoter sequence located at its 3′ end. The nucleicacid construct comprising the adequate insertion is selected by thedeletion of the nucleotide sequence 1 encoding poison 1 in a strainsensitive to it but resistant to poison 2. The deletion of the targetsequence (DNA fragment A) for re-use of the building block is achievedthrough the insertion of the DNA fragment initially removed (i.e.,nucleotide sequence 1 encoding poison 1 with a promoter at its 5′ end).This reverse event is selected in a strain sensitive to poison 2 andresistant to poison 1. Plasmid 1 is amplified in a strain resistant topoison 1. Plasmid 2 is amplified in a strain resistant to poison 2.

In FIG. 3, the insertion of the target sequence (DNA fragment A) isselected by the deletion of the nucleotide sequence 1 encoding poison 1in a strain sensitive to it. The deletion of the DNA fragment A forre-use of the building block is achieved through the insertion of theDNA fragment initially removed (i.e., poison 1 with, at its 5′ end, twopromoters in opposite directions). This reverse event is selected in astrain allowing the conditional expression of poison 2, sensitive topoison 2, and resistant to poison 1. Plasmid 1 is amplified in a strainresistant to poison 1. Plasmid 2 is amplified in any strain whoseviability is independent from the presence or absence of plasmid 2.

In FIG. 4, Plasmid 1 encodes both poison 1 and antidote 2 that areorganized as an operon. The insertion of the target sequence (DNAfragment A) is selected by the deletion of the nucleotide sequenceencoding poison 1 in a strain sensitive to it. The deletion of thetarget sequence (DNA fragment A) for re-use of the building block isachieved through the insertion of the DNA fragment initially removed(i.e., nucleotide sequence encoding poison 1 with a promoter at its 5′end). This reverse event is selected through the activation of thenucleotide sequence encoding antidote 2 in a strain allowing theconditional expression of poison 2, sensitive to poison 2, and resistantto poison 1. Plasmid 1 is amplified in a strain resistant to poison 1.Plasmid 2 is amplified in any strain whose viability is independent fromthe presence or absence of plasmid 2.

In FIG. 5, the target sequence (DNA fragment A) contains a promoterallowing the production of an antidote. The inversion of the DNAfragment A is selected using a strain allowing the conditionalexpression of poison 2 and sensitive to it. The reverse event isselected in a strain allowing the conditional expression of poison 1 andsensitive to it.

In other words, constructs produced through the invention are notdead-end products (i.e., useful for the only use they have been producedfor); they can be recycled. This emphasizes the importance of thesoftware component of the invention because it allows to create not onlya data base of building blocks, but also of products that are followedup and stored (virtually in computers, and physically in freezers orother devices) for potential future uses. Because the software tracksthe features of each building block and product, it also identifiesthose elements that are (i) necessary and (ii) inter-compatible forfuture and new multitask/parallel/reversible processes.

1. A genetic construct which is suitable for an insertion/deletion andan inversion for at least one target nucleotide sequence comprising: apromoter/activator sequence disposed upstream of a first nucleotidesequence encoding a first toxic molecule and a second nucleotidesequence encoding a second toxic molecule different from said firsttoxic molecule, or a first promoter/activator sequence disposed upstreamof the first nucleotide sequence encoding a first toxic molecule anddisposed in an opposite direction of said first nucleotide sequence, anda second promoter/activator sequence disposed upstream of a secondnucleotide sequence encoding an antidote to a second toxic moleculedifferent from said first toxic molecule, or a promoter/activatorsequence disposed upstream of a first nucleotide sequence encoding thefirst toxic molecule and a second nucleotide sequence encoding anantidote to the second toxic molecule different from said first toxicmolecule.
 2. The genetic construct according to claim 1 suitable for theinversion of at least one target nucleotide sequence and comprised of afirst promoter/activator sequence disposed upstream, of a firstnucleotide sequence encoding the toxic molecule, a second nucleotidesequence encoding an antidote to a second toxic molecule different fromsaid first toxic molecule wherein the second nucleotide sequence isdisposed in an opposite direction to the reading orientation of thefirst promoter/activator sequence, and, a third nucleotide sequenceencoding an antidote to said first toxic molecule.
 3. The geneticconstruct according to claim 1, wherein each nucleotide sequenceencoding a toxic molecule or an antidote to a toxic molecule is anucleic acid sequence which encodes a fusion protein active as a toxicmolecule or as an antidote to said toxic molecule, said nucleic acidsequence which encodes said fusion protein comprising several uniquecloning sites and a nucleotide sequence encoding a molecule toxic to acell or an antidote to said toxic molecule.
 4. The genetic constructaccording to claim 1, which further comprises recombination sitesdisposed upstream and downstream the nucleotide sequence(s) encoding thefirst and the second toxic molecules and/or the nucleotide sequence(s)encoding an antidote to a toxic molecule.
 5. The genetic constructaccording to claim 1, wherein the sequences encoding the toxic moleculeand the antidote to the toxic molecule are poison/antidote sequences. 6.A cloning vector comprising at least one of the genetic constructaccording to claim
 1. 7. The cloning vector according to claim 6 furthercomprising an origin of replication and a selectable marker.
 8. A celltransformed by the genetic construct of claim
 1. 9. The cell accordingto claim 8 which is selected from the group consisting of prokaryotecells, plant cells, animal cells and fungi cells.
 10. A cloning andselection kit comprising an element selected from the group consistingof one or more nucleic acid construct according to claim 1, one or morevectors comprising at least one genetic construct of claim 1 and cellsto be transformed by said construct or vector, wherein said cells areeither resistant or sensitive to one or more of said toxic molecule(s),or wherein said cells are expressing one or more of said toxicmolecule(s) or antidote(s) to said toxic molecule(s).
 11. A method foran insertion and/or an inversion of a target nucleotide sequence into anucleic acid construct said method comprising the following steps:providing a nucleic acid construct according to claim 1 and obtainingthe insertion of said target nucleotide sequence into the nucleic acidconstruct by inactivation of a nucleotide sequence encoding a toxicmolecule and selecting the nucleic acid construct having integrated saidtarget nucleotide sequence in a cell which is sensitive to said toxicmolecule.
 12. The method according to claim 11 which further comprisesthe step of replacing the target nucleotide sequence by elements whichhave been deleted following the insertion of said target nucleotidesequence or by the integration of a target nucleotide sequence having aninverted reading orientation.
 13. The method according to claim 12,wherein said integration of the target nucleotide sequence, replacementor inversion of the target nucleotide sequence is a step selected fromthe group consisting of restriction/ligation, site specificrecombination, TOPO cloning and homologous recombination.
 14. The methodaccording to claim 13, which comprises the step of insertion/deletionand/or reversion of several target nucleotide sequences into multiplenucleic acid construct(s) and the step of selecting simultaneously aconstruct having integrated, deleted or inverted said target nucleotidesequences.
 15. A computer program comprising program codes means forperforming the steps according to claim
 11. 16. A computer programproduct comprising the program codes means on a computer readable mediumfor performing the steps of the method according to claim 11 when saidprogram is run on a computer.
 17. An automate connected to a database ofa computer and which comprises an element selected from the groupconsisting of the genetic construct according to claim 1, a vectorcomprising at least one genetic construct of claim 1, and a celltransformed by the genetic construct of claim
 1. 18. The geneticconstruct of claim 2, wherein said second nucleotide sequence encodingsaid antidote to said second toxic molecule is under the control of asecond promoter/activator sequence.
 19. The genetic construct accordingto the claim 5, wherein the poison/antidote sequences are selected fromthe group consisting of the following poison/antidote systems:CcdB/CcdA, Kid/Kis, Hok/Sok, Doc/Phd, RelE/RelB, PasA/PasB/PasC,MazE/MazF and ParE/ParD.
 20. The vector of claim 7, wherein theselectable marker is an antibiotic resistance selectable marker.
 21. Thecell of claim 9, wherein said animal cells are human cells.
 22. The cellof claim 9, wherein said fungi cells are yeast cells.
 23. The method ofclaim 11 further comprising: selecting the target nucleotide sequencefrom genome databases through analysis of a corresponding genomicsequence by identification of exon-intron-structure and comparison withexpression genetic databases, providing primer sequences suitable for agenetic amplification and cloning of said target genetic sequence,selecting elements of the nucleic acid construct presented in databasesas well as cells to be transformed by the nucleic acid construct, andproviding a design of the nucleic acid construct suitable for anintegration of the target nucleotide sequence.
 24. The method accordingto claim 14, wherein the step of selecting simultaneously a constructhaving integrated, deleted or inverted the target nucleotide sequencesis made in a single cell or in a single reaction tube.